Vaccines have proven to be successful, highly acceptable methods for the prevention of infectious diseases. They are cost effective, and do not induce antibiotic resistance to the target pathogen or affect normal flora present in the host. In many cases, such as when inducing antiviral immunity, vaccines can prevent a disease for which there are no viable curative or ameliorative treatments available.
Vaccines function by triggering the immune system to mount a response to an agent, or antigen, typically an infectious organism or a portion thereof that is introduced into the body in a non-infectious or non-pathogenic form. Once the immune system has been xe2x80x9cprimedxe2x80x9d or sensitized to the organism, later exposure of the immune system to this organism as an infectious pathogen results in a rapid and robust immune response that destroys the pathogen before it can multiply and infect enough cells in the host organism to cause disease symptoms.
The agent, or antigen, used to prime the immune system can be the entire organism in a less infectious state, known as an attenuated organism, or in some cases, components of the organism such as carbohydrates, proteins or peptides representing various structural components of the organism, or nucleic acids encoding such components.
In many cases, it is necessary to enhance the immune response to the antigens present in a vaccine in order to stimulate the immune system to a sufficient extent to make a vaccine effective, i.e., to confer immunity. Many protein and most peptide and carbohydrate antigens, administered alone, do not elicit a sufficient antibody response to confer immunity. Such antigens need to be presented to the immune system in such a way that they will be recognized as foreign and will elicit an immune response. To this end, additives (adjuvants) have been devised which enhance the immune response.
The best known adjuvant, Freund""s complete adjuvant, consists of a mixture of mycobacteria in an oil/water emulsion. Freund""s adjuvant works in two ways: first, by enhancing cell and humoral-mediated immunity, and second, by blocking rapid dispersal of the antigen challenge (the xe2x80x9cdepot effectxe2x80x9d). However, due to frequent toxic physiological and immunological reactions to this material, Freund""s adjuvant cannot be used in humans.
Another molecule that has been shown to have immunostimulatory or adjuvant activity is endotoxin, also known as lipopolysaccharide (LPS). LPS stimulates the immune system by triggering an xe2x80x9cinnatexe2x80x9d immune responsexe2x80x94a response that has evolved to enable an organism to recognize endotoxin (and the invading bacteria of which it is a component) without the need for the organism to have been previously exposed. While LPS is too toxic to be a viable adjuvant, molecules that are structurally related to endotoxin, such as monophosphoryl lipid A (xe2x80x9cMPLxe2x80x9d) are being tested as adjuvants in clinical trials. Currently, however, the only FDA-approved adjuvant for use in humans is aluminum salts (Alum) which are used to xe2x80x9cdepotxe2x80x9d antigens by precipitation of the antigens. Alum also stimulates the immune response to antigens.
Thus, there is a recognized need in the art for compounds which can be co-administered with antigens in order to stimulate the immune system to generate a more robust response to the antigen than would be seen if the antigen were injected alone or with Alum.
In one aspect, the invention provides novel compounds that are capable of enhancing an immune response in an animal when administered to the animal. In one embodiment, the compounds of the invention function as immunological adjuvants when co-administered with antigens, including antigens used as vaccines for any disease or condition amenable to vaccination. The novel adjuvant compounds of the invention have the formula I. 
wherein
R1 is selected from the group consisting of
(a) xe2x80x94C(O)xe2x80x94;
(b) xe2x80x94C(O)xe2x80x94C1-14 alkylene-C(O)xe2x80x94 or xe2x80x94C(O)xe2x80x94C1-14 alkenylene-C(O)xe2x80x94, wherein the C1-14 alkylene or C1-14 alkenylene is optionally substituted with hydroxy, C1-6 alkoxy, C1-6 alkylenedioxy, carboxy, C1-6 alkoxycarbonyl, C1-6 carbamoyl, C1-6 acylamino, C1-6 alkylamino, or (aryl)C1-6 alkyl, wherein said aryl moiety of said (aryl)C1-6 alkyl is optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, (C1-6 alkoxy)C1-6 alkylamino, (C1-6 alkylamino)C1-6 alkoxy, xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C1-6alkylene-Oxe2x80x94C1-6 alkyl, xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C(O)xe2x80x94C1-6 alkylene-C(O)OH, or xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C(O)xe2x80x94C1-6 alkylene-C(O)xe2x80x94C1-6 alkyl;
(c) C2 to C15 straight or branched chain alkyl optionally substituted with hydroxy or alkoxy; and
(d) xe2x80x94C(O)xe2x80x94C6-12 arylene-C(O)xe2x80x94 wherein said arylene is optionally substituted with C1-6 alkyl, hydroxy, C1-6 alkoxy, halogen, nitro or amino;
a and b are independently an integer from 0 to about 4;
d and e are independently an integer from 1 to about 6;
dxe2x80x2 and exe2x80x2 are independently an integer from 0 to about 2;
X1 and Y1 are independently selected from the group consisting of a null, oxygen, xe2x80x94NHxe2x80x94, xe2x80x94N(C(O)C1-4 alkyl)-, and xe2x80x94N(C1-4 alkyl)-;
G1, G2, G3, and G4 are independently selected from the group consisting of oxygen, methylene, xe2x80x94NHxe2x80x94, xe2x80x94N(C1-4 alkyl)-, xe2x80x94N[C(O)xe2x80x94C1-4 alkyl]-, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94SO2xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94C(O)NHxe2x80x94, C(O)N(C1-4 alkyl), and xe2x80x94S(O)nxe2x80x94, where n is 0, 1, or 2;
R2, R3, R4, R5, R6, and R7 are independently selected from the group consisting of:
(a) C1 to C20 straight chain or branched chain alkyl which is optionally substituted with halo, oxo, hydroxy or alkoxy;
(b) C2 to C20 straight chain or branched chain alkenyl, alkynyl, or dialkenyl which is optionally substituted with halo, oxo, hydroxy or alkoxy; and 
xe2x80x83wherein
R8 is C1-6 straight or branched chain alkyl or C2-6 straight or branched chain alkenyl, alkynyl, or dialkenyl;
G5 is selected from the group consisting of oxygen, methylene, arylene, xe2x80x94NHxe2x80x94, xe2x80x94N(C1-4 alkyl)-, xe2x80x94N(C(O)xe2x80x94C1-4 alkyl)-, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94SO2xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94, and xe2x80x94S(O)nxe2x80x94, where n is 0, 1, or 2;
R9 and R10 are independently selected from the group consisting of
(i) C1 to C20 straight chain or branched chain alkyl which is optionally substituted with halo, oxo, hydroxy or alkoxy; and
(ii) C2 to C20 straight chain or branched chain alkenyl, alkynyl, or dialkenyl which is optionally substituted with halo, oxo, hydroxy or alkoxy;
xe2x80x83or any one or two of G1R2, G2R4, G3R5, and G4R7 may together be a hydrogen atom or hydroxyl;
or a pharmaceutically acceptable salt thereof.
In a second aspect, the present invention is directed to novel immunological formulations which comprise at least one of the adjuvant compounds of the invention.
In a third aspect, the invention is directed to novel immunological compositions which comprise an antigen and at least one of the adjuvant compounds of the invention.
In another aspect, the present invention is directed to methods for enhancing an immune response in an animal, comprising administering to the animal a compound of the invention.
The invention provides novel compounds that are capable of enhancing an immune response in an animal when administered to the animal. In certain preferred embodiments, the compounds of the invention are capable of producing in immunological effect when administered alone. In certain other preferred embodiments, the compounds of the invention function as immunological adjuvants when co-administered with antigens, including antigens used as vaccines for any disease or condition amenable to vaccination. The invention also provides immunological compositions comprising the novel compounds of the invention and methods for immunizing humans and non-human animals.
The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. The issued patents, applications, and references that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure will prevail.
For purposes of the present invention, the following definitions will be used:
Definitions
The term xe2x80x9cimmunological compositionxe2x80x9d, as used herein, is intended to include compositions capable of producing any effect on the immune system of an animal, including, without limitation, an immunoprophylactic, immunotherapeutic, immunopotentiating, or immunosuppressive effect.
The term xe2x80x9canimalxe2x80x9d, as used herein, refers to human patients and non-human animals. Non-human animals include any animal that is capable of mounting an immune response to a vaccine.
The terms xe2x80x9ccarbonylxe2x80x9d and xe2x80x9coxoxe2x80x9d, as used herein, refer to a (Cxe2x95x90O) moiety. A carbonyl group may also be represented as xe2x80x94C(O)xe2x80x94.
The term xe2x80x9cdicarbonylxe2x80x9d, as used herein, refers to a moiety with the structure xe2x80x94C(O)-alkylene-C(O)xe2x80x94 or xe2x80x94C(O)-arylene-C(O)xe2x80x94, which is bonded to a molecule through the carbon atoms of both terminal carbonyl moieties.
An xe2x80x9calkyl esterxe2x80x9d, as used herein, is a moiety with the structure xe2x80x94Oxe2x80x94C(O)-alkyl, which is bonded to a molecule through the singly bonded oxygen of the ester group. The term xe2x80x9calkoxycarbonylxe2x80x9d refers to a moiety with the structure xe2x80x94(O)xe2x80x94O-alkyl, which is bonded to a molecule through the carbonyl carbon atom.
An xe2x80x9calkenyl esterxe2x80x9d, as used herein, is a moiety with the structure xe2x80x94Oxe2x80x94C(O)xe2x80x94 carbon chain, where the carbon chain contains a carbon-to-carbon double bond, wherein the ester moiety is bonded to a molecule through its singly bonded oxygen atom.
The term xe2x80x9calkylenexe2x80x9d means a bivalent straight chain or branched alkyl hydrocarbon group.
The term xe2x80x9calkenylenexe2x80x9d means a bivalent straight chain or branched hydrocarbon group having at least one carbon to carbon double bond.
The term xe2x80x9cdialkenylenexe2x80x9d means a bivalent unsaturated straight chain or branched chain hydrocarbon group having at least two carbon to carbon double bonds.
The term xe2x80x9carylenexe2x80x9d refers to a bivalent aromatic group.
Where the terms xe2x80x9calkylenexe2x80x9d, xe2x80x9calkenylenexe2x80x9d, or xe2x80x9cdialkenylenexe2x80x9d include a descriptor indicating the number of carbon atoms or a range in the number of carbon atoms, e.g., C1-14 alkylene, the number of carbon atoms refers to the length of the carbon chain connecting the two chemical groups between which the alkylene group is positioned. When the term xe2x80x9carylenexe2x80x9d includes a descriptor indicating the number of carbon atoms or a range in the number of carbon atoms, the number of carbon i atoms refers to the number of carbon atoms in the aromatic ring system. Any of the carbon atoms of an alkylene, alkenylene, dialkenylene, or arylene group may be optionally substituted, as described below, and the substituents may contain additional carbon atoms.
The term xe2x80x9calkylxe2x80x9d as employed herein refers to straight and branched chain aliphatic groups having from 1 to 20 carbon atoms, which may be optionally substituted with one, two or three substituents.
An xe2x80x9carylxe2x80x9d group is a C6-C14 aromatic moiety comprising one to three aromatic rings, which may be optionally substituted. Preferably, the aryl group is a C6-C10 aryl group. The term xe2x80x9carylxe2x80x9d is also intended to include heteroaryl groups, comprising 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14xcfx80 electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to about three heteroatoms selected from the group consisting of N, O, and S.
An xe2x80x9caralkylxe2x80x9d or xe2x80x9carylalkylxe2x80x9d group comprises an aryl group, as defined above, which is covalently linked to an alkyl group, either of which independently may be optionally substituted or unsubstituted.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as employed herein refers to chlorine, bromine, fluorine, or iodine.
The term xe2x80x9cacylaminoxe2x80x9d refers to an amide group attached at the nitrogen atom. The term xe2x80x9ccarbamoylxe2x80x9d refers to an amide group attached at the carbonyl carbon atom. The nitrogen atom of an acylamino or carbamoyl substituent may be additionally substituted.
Unless otherwise explicitly limited, the term xe2x80x9caminoxe2x80x9d is meant to include NH2, alkylamino, dialkylamino, arylamino, aralkylamino, and cyclic amino groups.
As herein employed, the term xe2x80x9cacylxe2x80x9d refers to an alkylcarbonyl or arylcarbonyl substituent.
The abbreviation xe2x80x9cBocxe2x80x9d as used herein refers to t-butyloxycarbonyl.
The term xe2x80x9cnullxe2x80x9d as used herein with reference to a given substituent means that the substituent is absent, and the chemical groups between which the substituent is positioned are directly attached to each other by way of a covalent chemical bond.
As used herein with reference to compounds and compositions of the invention, the term xe2x80x9ctype 1xe2x80x9d refers to those compounds of the invention corresponding to formula I above where the values of a and b are the same; the values of d and e are the same; the values of dxe2x80x2 and exe2x80x2 are the same; X1 and Y1 are the same; G1 and G3 are the same; G2 and G4 are the same; R2 and R5 are the same; R3 and R6 are the same; and R4 and R7 are the same.
The term xe2x80x9ctype 2xe2x80x9d, as used herein, refers to compounds or compositions corresponding to formula I where any one or more of the following applies: the values of a and b are the different; the values of d and e are the different; the values of dxe2x80x2 and exe2x80x2 are the different; X1 and Y1 are the different; G1 and G3 are the different; G2 and G4 are the different; R2 and R5 are the different; R3 and R6 are the different; and R4 and R7 are the different.
Compounds
In a first aspect, the present invention provides novel compounds of formula I: 
wherein
R1 is selected from the group consisting of
(a) xe2x80x94C(O)xe2x80x94;
(b) xe2x80x94C(O)xe2x80x94C1-14 alkylene-C(O)xe2x80x94 or xe2x80x94C(O)xe2x80x94C1-14 alkenylene-C(O)xe2x80x94, wherein the C1-14 alkylene or C1-14 alkenylene is optionally substituted with hydroxy, C1-6 alkoxy, C1-6 alkylenedioxy, carboxy, C1-6 alkoxycarbonyl, C1-6 carbamoyl, C1-6 acylamino, C1-6 alkylamino, or (aryl)C1-6 alkyl, wherein said aryl moiety of said (aryl)C1-6 alkyl is optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, (C1-6 alkoxy)C1-6 alkylamino, (C1-6 alkylamino)C1-6 alkoxy, xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C1-6alkylenexe2x80x94Oxe2x80x94C1-6 alkyl, xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C(O)xe2x80x94C1-6 alkylene-C(O)OH, or xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C(O)xe2x80x94C1-6 alkylene-(O)xe2x80x94C1-6 alkyl;
(c) C2 to C15 straight or branched chain alkyl optionally substituted with hydroxy or alkoxy; and
(d) xe2x80x94C(O)xe2x80x94C6-12 arylene-C(O)xe2x80x94 wherein said arylene is optionally substituted with C1-6 alkyl, hydroxy, C1-6 alkoxy, halogen, nitro or amino;
a and b are independently an integer from 0 to about 4;
d and e are independently an integer from 1 to about 6;
dxe2x80x2 and exe2x80x2 are independently an integer from 0 to about 2;
X1 and Y1 are independently selected from the group consisting of a null, oxygen, xe2x80x94NHxe2x80x94, xe2x80x94N(C(O)C1-4 alkyl)-, and xe2x80x94N(C1-4 alkyl)-,
G1, G2, G3, and G4 are independently selected from the group consisting of oxygen, methylene, xe2x80x94NHxe2x80x94, xe2x80x94N(C1-4 alkyl)-, xe2x80x94N[C(O)xe2x80x94C1-4 alkyl]-, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94SO2xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94C(O)NHxe2x80x94, C(O)N(C1-4 alkyl), and xe2x80x94S(O)nxe2x80x94, where n is 0, 1, or 2;
R2, R3, R4, R5, R6, and R7 are independently selected from the group consisting of:
(a) C1 to C20 straight chain or branched chain alkyl which is optionally substituted with halo, oxo, hydroxy or alkoxy;
(b) C2 to C20 straight chain or branched chain alkenyl, alkynyl, or dialkenyl which is optionally substituted with halo, oxo, hydroxy or alkoxy; and 
xe2x80x83wherein
R8 is C1-6 straight or branched chain alkyl or C2-6 straight or branched chain alkenyl, alkynyl, or dialkenyl;
G3 is selected from the group consisting of oxygen, methylene, arylene, xe2x80x94NHxe2x80x94, xe2x80x94N(C1-4 alkyl)-, xe2x80x94N(C(O)xe2x80x94C1-4 alkyl)-, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94SO2xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94, and xe2x80x94S(O)nxe2x80x94, where n is 0, 1, or 2;
R9 and R10 are independently selected from the group consisting of
(i) C1 to C20 straight chain or branched chain alkyl which is optionally substituted with halo, oxo, hydroxy or alkoxy; and
(ii) C2 to C20 straight chain or branched chain alkenyl, alkynyl, or dialkenyl which is optionally substituted with halo, oxo, hydroxy or alkoxy;
xe2x80x83or any one or two of G1R2, G2R4, G3R5, and G4R7 may together be a hydrogen atom or hydroxyl;
or a pharmaceutically acceptable salt thereof.
The compounds of the invention are acidic, and are typically isolated in a corresponding salt form. Accordingly, specifically contemplated within the scope of the invention are compounds of Formula II: 
wherein M is a pharmaceutically acceptable cation, and all other variables are as defined above for Formula I. For divalent cations, the cation takes the place of two M variables in formula II above. Pharmaceutically acceptable cations are well known to those of ordinary skill in the art.
In some preferred embodiments of the invention, one or more of the following limitations is present: each of a and b is 2; each of X1 and Y1 is NH; each of d and e is 1 or 2; and each of dxe2x80x2 and exe2x80x2 is 0, 1, or 2. In certain preferred embodiments, each of d and e is 1 and each of dxe2x80x2 and exe2x80x2 is 0. In certain other preferred embodiments, each of and e is 1 and each of dxe2x80x2 and exe2x80x2 is 1 or 2.
In some preferred embodiments, R1 is xe2x80x94C(O)xe2x80x94 or xe2x80x94C(O)xe2x80x94C1-14 alkylene-C(O)xe2x80x94, wherein the C1-14 alkylene is optionally substituted with one or two substituents selected from the group consisting of hydroxy, C1-6 alkoxy, C1-6 alkylenedioxy, C1-6 alkylamino, or (aryl)C1-6 alkyl, wherein said aryl moiety of said (aryl)C1-6 alkyl is optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, (C1-6 alkoxy)C1-6 alkylamino, (C1-6 alkylamino)C1-6 alkoxy, xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C1-6alkylene-Oxe2x80x94C1-6 alkyl, xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C(O)xe2x80x94C1-6 alkylene-C(O)OH, or xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C(O)xe2x80x94C1-6 alkylene-C(O)xe2x80x94C1-6 alkyl.
In some preferred embodiments, G1, G2, G3, and G4 are each independently selected from the group consisting of xe2x80x94NHxe2x80x94C(O)xe2x80x94 and xe2x80x94Oxe2x80x94C(O)xe2x80x94.
In some preferred embodiments, at least two of R2-R7, R9, and R10 are C6-20 straight or branched chain alkyl, alkenyl, alkynyl, or dialkenyl, any of which groups may be optionally substituted with one or two substituents selected from the group consisting of halo, oxo, hydroxy and alkoxy. In certain preferred embodiments, at least two of R2-R7, R9, and R10 are C8-15 straight or branched chain alkyl, alkenyl, alkynyl, or dialkenyl, any of which groups may be optionally substituted with one or two substituents selected from the group consisting of halo, oxo, hydroxy and alkoxy.
In some preferred embodiments, at least four of R2-R7, R9, and R10 are C6-20 straight or branched chain alkyl, alkenyl, alkynyl, or dialkenyl, any of which groups may be optionally substituted with one or two substituents selected from the group consisting of halo, oxo, hydroxy and alkoxy. In certain preferred embodiments, at least four of R2-R7, R9, and R10 are C8-15 straight or branched chain alkyl, alkenyl, alkynyl, or dialkenyl, any of which groups may be optionally substituted with one or two substituents selected from the group consisting of halo, oxo, hydroxy and alkoxy.
In some preferred embodiments, at least six of R2-R7, R9, and R10 are C6-20 straight or branched chain alkyl, alkenyl, alkynyl, or dialkenyl, any of which groups may be optionally substituted with one or two substituents selected from the group consisting of halo, oxo, hydroxy and alkoxy. In certain preferred embodiments, at least six of R2-R7, R9, and R10 are C8-15 straight or branched chain alkyl, alkenyl, alkynyl, or dialkenyl, any of which groups may be optionally substituted with one or two substituents selected from the group consisting of halo, oxo, hydroxy and alkoxy.
Synthetic Methods
Type 1 Compounds
Type 1 compounds of Formula I, wherein d and e are each 1 and dxe2x80x2 and exe2x80x2 are each 0, are preferably synthesized according to the synthetic route depicted in Scheme 1. Thus, the aldehyde III, which is obtained from L-serine by literature procedures, is treated with an organometallic reagent such as a Grignard reagent to afford the alcohol IV. Acid treatment of IV, e.g., treatment with anhydrous HCl gas in methanol, affords the corresponding amino diol, which is selectively acylated on nitrogen to afford V. Protection of the primary alcohol, followed by acylation of the secondary alcohol then affords the compound VI. Deprotection of the primary alcohol and reaction with the phosphorylating reagent, prepared as described in the Experimental Section, then affords VII. Treatment with triethylsilane and trifluoroacetic acid effects removal of the Boc protecting group, and treatment with phosgene then affords the symmetrical dimer VIII. Finally, treatment with phenylsilane and tetrakis(triphenylphosphine)palladium(0) effects removal of the allyl protecting group to afford IX. 
Alternatively, intermediate IV may be prepared as a single diastereomer, preferably according to the synthetic route outlined in Scheme 2. 
Thus, aldehyde III is treated with allyl chloride and (+)-B-methoxydiisopino-campheylborane to afford X as a single diastereomer. Epoxide ring closure is effected by treatment with DBU, followed by SN2xe2x80x2 ring opening with an organocuprate reagent to afford XII. Hydrogenation then affords the alcohol XIII, which is converted to IX as described above for IV.
Type 1 compounds of Formula I, wherein d and e are each 1 and dxe2x80x2 and exe2x80x2 are each 1, are preferably prepared by procedures analogous to those outlined in Schemes 1 and 2 above, but starting with aldehyde XIV in place of III. Aldehyde XIV is preferably prepared by homologation of III, as outlined in Scheme 3. Thus, aldehyde III is treated with [2-(trimethylsilyl)ethoxymethyl]triphenylphosphonium chloride and n-butyllithium to afford the enol ether, which is treated with acetic acid to provide XIV. 
Type 1 compounds of Formula I with different values for d and e and/or dxe2x80x2 and exe2x80x2 are preferably prepared by methods generally analogous to those described above, as further described in the examples. One of skill in the art will also easily recognize that the synthetic methods outlined above may be adapted to the synthesis of compounds of Formula I, wherein G3 and/or G4 are other than xe2x80x94Oxe2x80x94C(O)xe2x80x94 or xe2x80x94NHxe2x80x94C(O)xe2x80x94, by performing alternative standard functional group transformations in place of the acylation steps described above.
Type 2 Compounds
Type 2 compounds of Formula I are preferably synthesized by methods analogous to those described for the synthesis of Type 1 compounds, up to the point just after cleavage of the protecting group from the primary amine group of the phosphate ester compound. At this point, the phosphate ester compound is treated with a monoprotected difunctionalized compound, such as a dicarboxylic acid. The resultant compound is deprotected and allowed to react with a second phosphate ester compound. Final phosphate deprotection is then accomplished as described for the Type 1 compounds.
An alternative method for the synthesis of the Type 2 compounds is to form the isocyanate intermediate of the deprotected primary amine group of the phosphate ester compound. The isocyanate intermediate is then treated with a second deprotected primary amine of a phosphate ester compound followed by deprotection as described for the Type 1 compounds.
Adjuvant and Vaccine Formulations and Administration
In a second aspect, the invention provides immunological compositions comprising a compound of formula I: 
wherein
R1 is selected from the group consisting of
(a) xe2x80x94C(O)xe2x80x94,
(b) xe2x80x94C(O)xe2x80x94C1-14 alkylene-C(O)xe2x80x94 or xe2x80x94C(O)xe2x80x94C1-14 alkenylene-C(O)xe2x80x94, wherein the C1-14 alkylene or C1-14 alkenylene is optionally substituted with hydroxy, C1-6 alkoxy, C1-6 alkylenedioxy, carboxy, C1-6 alkoxycarbonyl, C1-6 carbamoyl, C1-6 acylamino, C1-6 alkylamino, or (aryl)C1-6 alkyl, wherein said aryl moiety of said (aryl)C1-6 alkyl is optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1≢alkylamino, (C1-6 alkoxy)C1-6 alkylamino, (C1-6 alkylamino)C1-6 alkoxy, xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C1-6alkylene-Oxe2x80x94C1-6 alkyl, xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C(O)xe2x80x94C1-6 alkylene-C(O)OH, or xe2x80x94Oxe2x80x94C1-6 alkylene-NHxe2x80x94C(O)xe2x80x94C1-6 alkylene-C(O)xe2x80x94C1-6 alkyl;
(c) C2 to C15 straight or branched chain alkyl optionally substituted with hydroxy or alkoxy; and
(d) xe2x80x94C(O)xe2x80x94C6-12 arylene-C(O)xe2x80x94 wherein said arylene is optionally substituted with C1-6 alkyl, hydroxy, C1-6 alkoxy, halogen, nitro or amino;
a and b are independently an integer from 0 to about 4;
d and e are independently an integer from 1 to about 6;
dxe2x80x2 and exe2x80x2 are independently an integer from 0 to about 2;
X1 and Y1 are independently selected from the group consisting of a null, oxygen, xe2x80x94NHxe2x80x94, xe2x80x94N(C(O)C1-4 alkyl)-, and xe2x80x94N(C1-4 alkyl)-,
G1, G2, G3, and G4 are independently selected from the group consisting of oxygen, methylene, xe2x80x94NHxe2x80x94, xe2x80x94N(C1-4 alkyl)-, xe2x80x94N[C(O)xe2x80x94C1-4 alkyl]-, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94SO2xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94C(O)NHxe2x80x94, C(O)N(C1-4 alkyl), and xe2x80x94S(O)nxe2x80x94, where n is 0, 1, or 2;
R2, R3, R4, R5, R6, and R7 are independently selected from the group consisting of:
(a) C1 to C20 straight chain or branched chain alkyl which is optionally substituted with halo, oxo, hydroxy or alkoxy;
(b) C2 to C20 straight chain or branched chain alkenyl, alkynyl, or dialkenyl which is optionally substituted with halo, oxo, hydroxy or alkoxy; and 
xe2x80x83wherein
R8 is C1-6 straight or branched chain alkyl or C2-6 straight or branched chain alkenyl, alkynyl, or dialkenyl;
G5 is selected from the group consisting of oxygen, methylene, arylene, xe2x80x94NHxe2x80x94, xe2x80x94N(C1-4 alkyl)-, xe2x80x94N(C(O)xe2x80x94C1-4 alkyl)-, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94SO2xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94, and xe2x80x94S(O)nxe2x80x94, where n is 0, 1, or 2;
R9 and R10 are independently selected from the group consisting of
(i) C1 to C20 straight chain or branched chain alkyl which is optionally substituted with halo, oxo, hydroxy or alkoxy; and
(ii) C2 to C20 straight chain or branched chain alkenyl, alkynyl, or dialkenyl which is optionally substituted with halo, oxo, hydroxy or alkoxy;
xe2x80x83or any one or two of G1R2, G2R4, G3R5, and G4R7 may together be a hydrogen atom or hydroxyl;
or a pharmaceutically acceptable salt thereof; and
a pharmaceutically acceptable carrier, diluent, or excipient.
Preferred embodiments according to this aspect of the invention are as described above for the first aspect.
The invention is also directed to novel immunological compositions comprising a compound of formula I, as described above; an antigen; and a pharmaceutically acceptable carrier, diluent, or excipient.
The immunological composition may utilize any suitable antigen or vaccine component in combination with an adjuvant compound of the invention. As a further example, such immunological compositions may suitably comprise an attenuated organism, or a component of an organism, such as a carbohydrate, protein, or peptide, or a nucleic acid encoding such component.
Typically, an antigen is employed in mixture with the adjuvant compounds of the invention. In certain other embodiments, it may be useful in some applications to employ an antigen covalently linked to an amino, carboxyl, hydroxyl and/or phosphate moiety of the adjuvant compounds of the invention. The specific formulation of therapeutically effective compositions of the present invention may thus be carried out in any suitable manner which will render the adjuvant bioavailable, safe and effective in the subject to whom the formulation is administered.
Such immunological compositions may, for example, comprise at least one antigenic agent used as a vaccine for any disease or condition amenable to vaccination, including, but not limited to:
(A) human and animal infectious diseases, including those caused by bacteria, viruses, parasites (e.g., mycoplasmas, fungi, protozoa) and prions;
(B) diseases or pathologies in which an immune response against an autologous molecule may be beneficial, such as, but not limited to, Alzheimer""s disease, in which immunization to amyloid xcex242 may be beneficial; gastric reflux disease, in which immunization to gastrin may be beneficial; cancer, including, without limitation, melanoma, prostate, and colon cancer, in which immunization to cancer antigens may be beneficial; and autoimmune disorders, including, without limitation, diabetes, in which immunization to insulin may decrease or redirect inflammatory responses against insulin-producing cells; and
(C) non-pathological situations in which an immune response may bring about a desired change in function or physiology, such as, but not limited to, the contraceptive effect induced by immunization to hCG.
As further examples, the immunological compositions of the invention may comprise antigens or vaccine components which are pharmacologically active for disease states and conditions such as smallpox, yellow fever, distemper, cholera, fowl pox, scarlet fever, diphtheria, tetanus, whooping cough, influenza, rabies, mumps, HIV, chicken pox, rubella, measles, foot and mouth disease, and poliomyelitis.
In the resulting vaccine formulation, comprising (i) an antigen, and (ii) at least one adjuvant compound of the invention, the antigen and adjuvant compound are each present in an amount effective to elicit an immune response when the formulation is administered to a host animal, embryo, or ovum vaccinated therewith.
In further embodiments, the compounds of the invention may be covalently bonded to vaccine antigens, for example through an amino, thiol, carboxyl, hydroxyl or phosphate moiety. Methods of linking the adjuvant compositions of the invention to vaccine antigens are understood by persons of ordinary skill in the art in view of this disclosure. The adjuvant compositions may be linked to vaccines by any of the methods described in P. Hoffman et al., Biol. Chem. Hoppe-Sayler, 1989, 370:575-582; K.-H. Wiesmuller et al., Vaccine, 1989, 7:29-33; K.-H Wiesmuller et al., Int. J. Peptide Protein Res., 1992, 40:255-260; J.-P. Defourt et al., Proc. Natl. Acad. Sci. 1992, 89:3879-3883; T. Tohokuni et al., J. Am. Chem. Soc., 1994, 116:395-396; F. Reichel, Chem. Commun., 1997, 2087-2088; H. Kamitakahara, Angew. Chem. Int. Ed. 1998, 37:1524-1528; W. Dullenkopf et al., Chem. Eur. J., 1999, 5:2432-2438; all of which are hereby incorporated by reference.
The resulting vaccine formulations, including (i) an antigen, and (ii) an adjuvant compound, are usefully employed to induce an immunological response in an animal, by administering to such animal the vaccine formulation, in an amount sufficient to produce an antibody response in such animal.
The modes of administration may comprise the use of any suitable means and/or methods for delivering the adjuvant, adjuvant-containing vaccine, or adjuvant and/or antigen to one or more corporeal loci of the host animal where the adjuvant and associated antigens are immumostimulatorily effective. Delivery modes may include, without limitation, parenteral administration methods, such as subcutaneous (SC) injection, transcutaneous, intranasal (IN), ophthalmic, transdermal, intramuscular (IM), intradermal (ID), intraperitoneal (IP), intravaginal, pulmonary, and rectal administration, as well as non-parenteral, e.g., oral, administration.
The dose rate and suitable dosage forms for the adjuvant and vaccine compositions of the present invention may be readily determined by those of ordinary skill in the art without undue experimentation, by use of conventional antibody titer determination techniques and conventional bioefficacy/biocompatibility protocols, and depending on the particular antigen or therapeutic agent employed with the adjuvant, the desired therapeutic effect, and the desired time span of bioactivity.
The adjuvant of the present invention may be usefully administered to the host animal with any other suitable pharmacologically or physiologically active agents, e.g., antigenic and/or other biologically active substances.
Formulations of the invention can include additional components such as saline, oil, squalene, oil-water dispersions, liposomes, and other adjuvants such as QS-21, muramyl peptides, Freund""s incomplete adjuvant, and the like.
The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.